Generation of a human iPSC line (MPIi008-A) from a patient with Denys-Drash syndrome.

An induced pluripotent stem cell (hiPSC) line (MPIi008-A) was generated from fibroblasts of a 1-year-old male patient with Denys-Drash syndrome using lentiviral delivery of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. The MPIi008-A iPSC line exhibited typical iPSC morphology and normal karyotype, expressed pluripotent stem cell markers, and showed developmental potential to differentiate into derivatives of all three germ layers in vivo. The hiPSC line harbours a heterozygous missense mutation (R394L) in exon 9 of the WT1 gene.

Role for first zinc finger of WT1 in DNA sequence specificity: Denys-Drash syndrome-associated WT1 mutant in ZF1 enhances affinity for a subset of WT1 binding sites.

Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1-4). An alternative splicing of Wt1 can add three additional amino acids-lysine (K), threonine (T) and serine (S)-between ZF3 and ZF4. In the -KTS isoform, ZF2-4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type -KTS isoform ZF1-4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2-4, ZF1 can be positioned continuously in the DNA major groove forming a 'near-cognate' complex. Second, while ZF2-4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys-Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.

Repression of CMIP transcription by WT1 is relevant to podocyte health.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

The role of VEGF 165b in pathophysiology.

Anti-angiogenic vascular endothelial growth factor A (VEGF) 165b and pro-angiogenic VEGF 165 are generated from the same transcript, and their relative amounts are dependent on alternative splicing. The role of VEGF 165b has not been investigated in as much detail as VEGF 165, although it appears to be highly expressed in non-angiogenic tissues and, in contrast with VEGF 165, is downregulated in tumors and other pathologies associated with abnormal neovascularization such as diabetic retinopathy or Denys Drash syndrome. VEGF 165b inhibits VEGFR2 signaling by inducing differential phosphorylation, and it can be used to block angiogenesis in in vivo models of tumorigenesis and angiogenesis-related eye disease. Recent reports have identified three serine/arginine-rich proteins, SRSF1, SRSF2 and SRSF6, and studied their role in regulating terminal splice-site selection. Since the balance of VEGF isoforms is lost in cancer and angiogenesis-related conditions, control of VEGF splicing could also be used as a basis for therapy in these diseases.

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.

Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.

A proteomic investigation of glomerular podocytes from a Denys-Drash syndrome patient with a mutation in the Wilms tumour suppressor gene WT1.

Glomerular podocytes are essential for blood filtration in the kidney underpinned by their unique cytoskeletal morphology. An increasing number of kidney diseases are being associated with key podocyte abnormalities. The Wilms tumour suppressor gene (WT1) encodes a zinc finger protein with a crucial role in normal kidney development; and in the adult, WT1 is required for normal podocyte function. Denys-Drash Syndrome (DDS) results from mutations affecting the zinc finger domain of WT1. The aim of this study was to undertake, for the first time, a proteomic analysis of cultured human podocytes; and to analyse the molecular changes in DDS podocytes. The morphology of DDS podocytes was highly irregular, reminiscent of a fibroblastic appearance. A reference 2-D gel was generated, and 75 proteins were identified of which 43% involved in cytoskeletal architecture. The DDS and wild-type proteomes were compared by 2-D DIGE. The level of 95.6% of proteins was unaltered; but 4.4% were altered more than two-fold. A sample of proteins involved in cytoskeletal architecture appeared to be misexpressed in DDS podocytes. Consistent with this finding, overall levels of filamentous actin also appeared reduced in DDS podocytes. We conclude that one of WT1 functions in podocytes is to regulate the expression of key components and regulators of the cytoskeleton.

Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome.

Individuals with Denys-Drash syndrome (DDS) develop diffuse mesangial sclerosis, ultimately leading to renal failure. The disease is caused by mutations that affect the zinc finger structure of the Wilms' tumor protein (WT1), but the mechanisms whereby these mutations result in glomerulosclerosis remain largely obscure. How WT1 regulates genes is likely to be complex, because it has multiple splice forms, binds both DNA and RNA, and associates with spliceosomes. Herein is described that in DDS podocytes, the ratio of both WT1 +KTS isoforms C to D differs considerably from that of normal child and adult control podocytes and more closely resembles fetal profiles. Aside from the delay in podocyte maturation, DDS glomeruli show swollen endothelial cells, reminiscent of endotheliosis, together with incompletely fused capillary basement membranes; a dramatic decrease in collagen alpha4(IV) and laminin beta2 chains; and the presence of immature or activated mesangial cells that express alpha-smooth muscle actin. Because appropriate vascular endothelial growth factor A (VEGF-A) expression is known to be essential for the development and maintenance of glomerular architecture and function, this article addresses the question of whether VEGF-A expression is deregulated in DDS. The data presented here show that DDS podocytes express high levels of the proangiogenic isoform VEGF165, but completely lack the inhibitory isoform VEGF165b. The VEGF165/VEGF165b ratio in DDS resembles that of fetal S-shaped bodies, rather than that of normal child or adult control subjects. The alteration in VEGF-A expression presented here may provide a mechanistic insight into the pathogenesis of DDS.

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.

The dysregulated glomerular cell growth in Denys-Drash syndrome.

While diffuse mesangial sclerosis is traditionally described as being the glomerulopathy of Denys-Drash syndrome (DDS), the podocyte proliferative lesions may be overlooked in these DDS cases. In the present study, an evolving process is extrapolated from a selected case of DDS that demonstrated glomerulopathy with conspicuous podocyte proliferation. The observation that podocytes express proliferation markers (Ki67, proliferating-cell nuclear antigen and topoisomerase IIalpha) in non-proliferative, mature-looking glomeruli suggests an initial pathogenic act to activate or to keep podocytes from quiescence. The subsequent proliferation of podocytes is in keeping with downregulation of WT1 and cyclin kinase inhibitors of p16 and p21. The emergence of cytokeratin-positive cells in glomeruli that show typical mesangial sclerosis implies elimination of podocytes and replacement with tubular and/or parietal epithelial cells. The final scene of evolving glomerulopathy displays apoptosis and expression of Fas-L and Bax in sclerotic mesangial lesions, which eventually end up with global sclerosis. This novel concept of DDS glomerulopathy implies complex molecular mechanisms involved in glomerular injury.

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1.

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice.

Gonad development in Drash and Frasier syndromes depends on WT1 mutations.

The study of the gonads of 8 cases of Drash syndrome (6 ambiguous males, 2 females) and of 2 Frasier syndrome shows that WT1 mutations gives a dysgenetic testis which is the cause of the genital ambiguity observed at birth. By contrast the same mutations have no effect on ovary development giving normal females. However intron mutations in KTS with isoforms imbalance of WT1 proteins cause streak gonads with a female phenotype in XY patients. In consequence WT1 mutations are the cause of a spectrum of male genital malformations associated with glomerulonephritis and tumors. The absence of WT1 protein detection in sertoli cells shown by immunohistochemistry for 3 cases suggests an imprinting effect of the normal WT1 allele promotor rather than a low level of protein production. A caryotype is mandatory for a correct diagnosis.

A mutant form of the Wilms' tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.

Gonadoblastoma and dysgerminoma associated with XY gonadal dysgenesis in an adolescent with chronic renal failure: a case of Frasier syndrome.

STUDY OBJECTIVES: To report a rare reason for primary amenorrhea, a Frasier syndrome, XY gonadal dysgenesis associated with renal failure with eventual development of gonadoblastoma. To study immunohistochemical analysis of gonadoblastoma and dysgerminoma. To analyze the possibility of androgen receptor mutation in this rare syndrome. METHODS: We report a case of a 16-yr-old female with this syndrome. She underwent a laparoscopic bilateral gonadectomy and salpingectomy. A histopathological examination revealed gonadoblastoma with focal malignant dysgerminoma in the left dysgenetic gonad and an immunohistochemical of these fairly rare, malignant tumors. An androgen receptor was coded. Analysis was done. RESULTS: Immunohistochemical analysis showed that inhibin was strongly positive in gonadoblastoma but negative in dysgerminoma. No mutations of the androgen receptor gene were found. CONCLUSIONS: Inhibin positivity in gonadal stroma and in gonadoblastoma may indicate hormonal activity causing advanced puberty in patients with XY gonadal dysgenesis.

An unusual phenotype of Frasier syndrome due to IVS9 +4C>T mutation in the WT1 gene: predominantly male ambiguous genitalia and absence of gonadal dysgenesis.

The Wilms' tumor gene (WT1) encodes a zinc-finger transcription factor involved in the development of the kidneys and gonads and their subsequent normal function. Mutations in the WT1 gene were identified in patients with WAGR (Wilms' tumor, aniridria, genitourinary abnormalities, and mental retardation), Denys-Drash syndrome, and Frasier syndrome (FS). Constitutional heterozygous mutations of the WT1 gene, almost all located at intron 9, are found in patients with FS. This syndrome is characterized by female external genitalia in 46,XY patients, late renal failure, streak gonads, and high risk of gonadoblastoma development. We report a male with FS with an unusual phenotype characterized by normal penis size with perineal hypospadias, end-stage renal failure at the age of 19 yr, normal adult male serum T levels, extremely elevated gonadotropin levels, para-testicular leiomyoma, unilateral testicular germ cell tumor, bilateral gonadoblastoma, and absence of gonadal dysgenesis. Automatic sequencing identified the IVS9 +4C>T mutation in the WT1 gene, which predicts a change in splice site utilization. WT1 transcript analysis showed reversal of the normal positive/negative KTS (lysine, threonine, and serine) isoform ratio, confirming the diagnosis of FS. This patient with FS presents an external genitalia of Denys-Drash syndrome, suggesting that these two syndromes are not distinct diseases but may represent two ends of a spectrum of disorders caused by alterations in WT1 gene. This case expands the spectrum of phenotypes associated with WT1 mutations, by including predominantly male ambiguous genitalia and absence of gonadal dysgenesis, extremely high gonadotropin levels, and delayed adrenarche, and presence of a para-testicular leiomyoma, bilateral gonadoblastoma, and germ cell neoplasia.

Zinc finger proteins as potential targets for toxic metal ions: differential effects on structure and function.

Zinc finger structures are frequently found in transcription factors and DNA repair proteins, mediating DNA-protein and protein-protein binding. As low concentrations of transition metal compounds, including those of cadmium, nickel, and cobalt, have been shown to interfere with DNA transcription and repair, several studies have been conducted to elucidate potential interactions of toxic metal ions with zinc-binding protein domains. Various effects have been identified, including the displacement of zinc, e.g., by cadmium or cobalt, the formation of mixed complexes, incomplete coordination of toxic metal ions, as well as the oxidation of cysteine residues within the metal-binding domain. Besides the number of cysteine and/or histidine ligands, unique structural features of the respective protein under investigation determine whether or not zinc finger structures are disrupted by one or more transition metals. As improper folding of zinc finger domains is mostly associated with the loss of correct protein function, disruption of zinc finger structures may result in interference with manifold cellular processes involved in gene expression, growth regulation, and maintenance of the genomic integrity.